Our long-term objectives are to understand the molecular basis of prestin function, to advance the field closer to designing therapeutics in certain types of hearing loss. Prestin, a membrane protein in outer hair cells in the cochlea, is involved in cochlear amplification leading to frequency sensitivity. Several lines of evidence indicate that prestin oligomerizes, but the functional significance of this is unclear. In this proposal, we aim to dissect the functional roles of prestin monomeric and oligomeric species. Towards this objective, we will pursue two Specific Aims. In Aim 1, we will use computational, mutagenesis, biochemical and electrophysiological techniques to identify and characterize the prestin-prestin interaction residues in the cytoplasmic domains of full-length prestin. In Aim 2, we will use biochemical and biophysical techniques on the isolated prestin cytoplasmic domains, combined with thermodynamic analysis to construct a model describing the functional contributions of various monomeric and oligomeric prestin species. Mechanical stimuli caused by sound waves are amplified within the cochlea, giving rise to sensitivity to a wide range of frequencies. This amplification is powered at least in part by a mechanical motor in specialized cells called outer hair cells (OHCs) in the cochlea. Prestin, a membrane protein in OHCs, was discovered in 2000 as an essential component of this motor. Mice that lack prestin show defects in hearing and certain types of hearing loss are associated with mutations in prestin. Knowledge of the basis of prestin function is therefore essential for understanding and treating certain types of hearing loss. In this project, we will use computational analyses to identify putative functional residues in prestin, and then use a combination of biological, biophysical and electrophysiological studies to identify and characterize the roles of specific residues that are essential for function. Our studies are comprehensive and systematic and will yield important information about prestin function that will aid in the design of therapeutics to certain types of hearing loss. PUBLIC HEALTH RELEVANCE: According to statistics from the NIDCD, approximately 15% (32.5 million) of Americans have a hearing impairment. Understanding the mechanistic basis of cochlear function is an important first step to treating the underlying causes of hearing loss. Our research proposal uses a combination of approaches to achieve this goal by investigating the molecular basis of function of prestin, an important cochlear component.